Research in my laboratory focuses on the understanding of the mechanisms of bacterial pathogenesis. Our current focus is on the gram-negative bacterium, Helicobacter pylori, that chronically infects a large percentage of the worlds population (50 to 90%) and is the causative agent for gastritis, ulcer disease and some gastric cancers. To date, the mechanism of H. pylori pathogenesis is not completely understood. In mice, we know that H. pylori can infect and survive in the stomach. In human, the infection can last a life-time suggesting that it can successfully evade the immune responses. Although previously thought to be an extracellular organism, H. pylori, in recently reported in vitro studies, may be a facultative intracellular bacterium. This observation may partially explain its immune and therapeutic evasion.[unreadable] [unreadable] Identification of the genes accounting for cell growth, stress responses and evasion of host immune responses is fundamental to understanding the mechanisms of pathogenesis.[unreadable] We are using C57BL/6 mice and the murine macrophage cell line, RAW 264.7 to screen for additional H. pylori virulence factors. Initial experiments involving H. pylori infection of macrophage and subsequent gentamicin treatment demonstrated that H. pylori can infect macrophage cells and can survive intracellularly for more than 24 hours. Fluorescence and confocal microscopic studies further confirmed the presence of H. pylori in these cells.[unreadable] [unreadable] In collaboration with Drs. Ding Jin and Yan Zhou, NCI, we tested whether the spoT gene of H. pylori is important for environmental stresses and intracellular survival. The response to stress by many pathogenic and nonpathogenic bacteria is mediated by increased intracellular levels of hyperphosphorylated guanine nucleotides, specifically the 3-pyrophosphate derivative of GDP (ppGpp) and the 3-pyrophosphate derivative of GTP (pppGpp). We constructed spoT null mutation in the Sydney strain 1 (SS1) and G27 H. pylori strains. We characterized spoT mutants of H. pylori and found that the spoT gene encodes a bifunctional enzyme that catalyzes both hydrolase and synthetase (p)ppGpp activities. The spoT null mutant grows to a higher density in serum free medium than a spoT wild-type strain, mimicking a relaxed phenotype. Further, we showed that in serum free medium, the morphological change from helical to non-helical shape was delayed in SpoT+ strains. SpoT is important for intracellular survival in macrophages following phagocytosis. [unreadable] [unreadable] With a genetic method known as in vivo expression technology (IVET), we probe microbial genes involved in pathogenicity. IVET is a practical strategy for identifying a subset of genes induced preferentially during infection of an animal host. We use an antibiotic-based variant of IVET involving random DNA fragments of H. pylori fused to a tandem-reporter system of chloramphenicol acetyltransferase (cat) and beta-galactosidase (lacZ). We constructed unique H. pylori promoter-screening vectors (pIVET11 and pIVET12). A H. pylori genomic bank constructed in pIVET11 or pIVET12 was transformed in H. pylori strain HP1061 to generate a library of merodiploid (co-integrated) strains. These strains are plasmid recombinants through homologous recombination into the HP1061 genome at different loci. We selected the 702 merodiploid strains containing fusions of the cat gene that are transcriptionally active in mouse stomachs or within macrophage. Through the IVET screening in mice and macrophage cell line RAW264.7, we identified 38 in vivo induced genes (ivi genes) and the previously reported virulence factors cagA and vacA. These 38 ivi genes fall under several functional categories: motility/chemotaxis, LPS, outer membrane proteins, protein synthesis, oxidative stress response, cell division, transposases, DNA modification, acid resistance, hypothetical proteins and pathogenesis. The expression levels, based on RT-PCR analysis, of a large number of the identified ivi genes are upregulated 2-20 fold in H. pylori that were phagocytized by RAW 264.7. We are testing several of these in vivo induced genes to define their roles in H. pylori infection, survival and pathogenesis.[unreadable] We have developed and validated the use of a H. pylori specific IVET variant to identify the ivi genes in mice and cultured macrophage.